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A method is described that allows simultaneous measurement of two spectrally distinguishable green fluorescent protein (GFP) mutants with a confocal microscope. In contrast to previously described methods, neither UV excitation nor repetition of scans is required. Therefore the method is well-suited to the long-time observation of living cells in three-dimensional microscopy and time series recording, as demonstrated with GFP-expressing Dictyostelium discoideum cells.